Petra van den Broek

Detection of contamination with antineoplastic agents in a hospital pharmacy department

The contamination with fluorouracil, cyclophosphamide and methotrexate was studied in a hospital pharmacy department where these drugs were prepared. In the preparation room, air samples were taken before and during preparation of the drugs. Methotrexate was detected in one sample which was collected during preparation (0.3μg/m3). Spot samples were taken in the vertical laminar airflow safety hood before and after preparation of the drugs and after cleaning of the hood. Contamination of the laminar airflow hood was: cyclophosphamide: 1–160 ng/cm2; fluorouracil: 10–62 ng/cm2 and methotrexate: 2–633 ng/cm2. Spot samples from the floor in front of and beneath the laminar airflow hood showed contamination with especially fluorouracil (48–236μg/m2). The gloves used during preparation of the drugs were contaminated mainly with fluorouracil (5–980 ng/cm2). Urine samples from two workers involved in the preparation of the drugs were analysed for unmetabolized cyclophosphamide; it was not detected. Although no uptake of cyclophosphamide was established, it is shown that the methods for measurement of cyclophosphamide, fluorouracil and methotrexate in the preparation room are applicable for the control of occupational exposure to these drugs.

Oral therapy with dipyridamole limits ischemia-reperfusion injury in humans

Adenosine receptor stimulation induces several effects that could limit ischemia‐reperfusion injury. We hypothesize that treatment with the nucleoside uptake inhibitor dipyridamole increases endogenous adenosine and limits ischemia‐reperfusion injury in humans.

Enhanced Cellular Adenosine Uptake Limits Adenosine Receptor Stimulation in Patients With Hyperhomocysteinemia

Objective— Endogenous adenosine has several cardioprotective effects. We postulate that in patients with hyperhomocysteinemia increased intracellular formation of S-adenosylhomocysteine decreases free intracellular adenosine. Subsequently, facilitated diffusion of extracellular adenosine into cells through dipyridamole-sensitive transporters is enhanced, limiting adenosine receptor stimulation. We tested this hypothesis in patients with classical homocystinuria (n=9, plasma homocysteine 93.1±24.7 &mgr;mol/L) and matched controls (n=8, homocysteine 9.1±1.0). Methods and Results— Infusion of adenosine (0.5, 1.5, 5.0, and 15.0 &mgr;g/min/dL forearm) into the brachial artery increased forearm blood flow, as measured with venous occlusion plethysmography, to 2.9±0.4, 4.3±0.5, 5.6±1.1, and 9.6±2.1 in the patients and to 2.8±0.6, 4.4±1.0, 9.0±1.7, and 17.0±3.1 mL/min/dL in controls (P<0.05). However, adenosine-induced vasodilation in the presence of dipyridamole (100 &mgr;g/min/dL) was similar in both groups (P=0.9). Additionally, in isolated erythrocytes, adenosine uptake was accelerated by incubation with homocysteine (half-time 6.4±0.3 versus 8.1±0.5 minutes, P<0.001) associated with increased intracellular formation of S-adenosylhomocysteine (P<0.0001). Conclusions— In hyperhomocysteinemia, adenosine-induced vasodilation is impaired but is restored by dipyridamole. Accelerated cellular adenosine uptake probably accounts for these observations. These impaired actions of adenosine could well contribute to the cardiovascular complications of hyperhomocysteinemia.

Role of multidrug resistance protein 2 (MRP2) in glutathione-bimane efflux from Caco-2 and rat renal proximal tubule cells.

The multidrug resistance protein 2 (MRP2) has been shown to play an important role in the transport of glutathione conjugates in the liver. Its importance in renal excretion, however, is still uncertain and other organic anion transporters may be involved. The objective of the present study was to characterize glutathione conjugate efflux from rat kidney proximal tubule cells (PTC), and to determine the contribution of Mrp2. We used isolated PTC in suspension, as well as grown to monolayer density. For comparison, transport characteristics were also determined in the human intestinal epithelial cell line Caco‐2, an established model to study MRP2‐mediated transport. The cells were loaded with monochlorobimane (MCB) at 10°C. MCB enters the cells by simple diffusion and is conjugated with glutathione to form the fluorescent glutathione‐bimane (GS‐B). In primary cultures of rat PTC, no indications for a transporter‐mediated mechanism were found. The efflux of GS‐B from Caco‐2 cells and freshly isolated PTC was time‐ and temperature‐dependent. Furthermore, GS‐B transport in both models was inhibited by chlorodinitrobenzene (CDNB), with an inhibitory constant of 46.8±0.9 μM in freshly isolated PTC. In Caco‐2 cells, the inhibitory potency of CDNB was approximately 20 fold higher. Finally, efflux of GS‐B from freshly isolated PTC from Mrp2‐deficient (TR−) rats was studied. As compared to normal rat PTC, transport characteristics were not different. We conclude that in freshly isolated rat PTC glutathione conjugate excretion is mediated by other organic anion transporters rather than by Mrp2.

The Influence of Dietary Protein Intake on Mammalian Tryptophan and Phenolic Metabolites

Although there has been increasing interest in the use of high protein diets, little is known about dietary protein related changes in the mammalian metabolome. We investigated the influence of protein intake on selected tryptophan and phenolic compounds, derived from both endogenous and colonic microbial metabolism. Furthermore, potential inter-species metabolic differences were studied. For this purpose, 29 healthy subjects were allocated to a high (n = 14) or low protein diet (n = 15) for 2 weeks. In addition, 20 wild-type FVB mice were randomized to a high protein or control diet for 21 days. Plasma and urine samples were analyzed with liquid chromatography–mass spectrometry for measurement of tryptophan and phenolic metabolites. In human subjects, we observed significant changes in plasma level and urinary excretion of indoxyl sulfate (P 0.004 and P 0.001), and in urinary excretion of indoxyl glucuronide (P 0.01), kynurenic acid (P 0.006) and quinolinic acid (P 0.02). In mice, significant differences were noted in plasma tryptophan (P 0.03), indole-3-acetic acid (P 0.02), p-cresyl glucuronide (P 0.03), phenyl sulfate (P 0.004) and phenylacetic acid (P 0.01). Thus, dietary protein intake affects plasma levels and generation of various mammalian metabolites, suggesting an influence on both endogenous and colonic microbial metabolism. Metabolite changes are dissimilar between human subjects and mice, pointing to inter-species metabolic differences with respect to protein intake.

Upregulation of ecto-5'-nucleotidase by rosuvastatin increases the vasodilator response to ischemia.

3-Hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors (statins) are effective in the primary and secondary prevention of cardiovascular events. Although originally developed to improve lipid profile, statins have demonstrated a surplus of beneficial pleiotropic effects, including improved endothelial function, reduced inflammation, and increased tolerance to ischemia-reperfusion injury. In preclinical studies, increased ecto-5′-nucleotidase activity, the key enzyme in extracellular adenosine formation, plays an important role in these effects. Because human data are absent, we explored the effects of rosuvastatin on ecto-5′-nucleotidase activity and the clinical relevance of increased extracellular adenosine during ischemia in humans in vivo. The forearm vasodilator responses to 3 increasing periods of forearm ischemia (2, 5, and 13 minutes) were determined during placebo and caffeine (an adenosine receptor antagonist) infusion into the brachial artery. At the end of an 8-day treatment period with rosuvastatin (20 mg per day), this whole procedure was repeated. During both experiments, ecto-5′-nucleotidase activity was determined. Vasodilator responses are expressed as the percentage increase in forearm blood flow ratio from baseline. Rosuvastatin increased ecto-5′-nucleotidase activity by 49±17% and enhanced the vasodilator response after 2, 5, and 13 minutes of ischemia in the absence (146±19, 330±26, and 987±133 to 312±77, 566±107, and 1533±267) but not in the presence of caffeine (98±25, 264±54, and 727±111 versus 95±19, 205±34, and 530±62). Rosuvastatin increases extracellular formation of adenosine in humans in vivo probably by enhancing ecto-5′-nucleotidase activity. This action results in the improvement of reactive hyperemia and may further enhance the clinical benefit of statins, in particular in conditions of ischemia.

The 22G>A polymorphism in the adenosine deaminase gene impairs catalytic function but does not affect reactive hyperaemia in humans in vivo.

Objectives During ischaemia, the extracellular concentration of the endogenous nucleoside adenosine increases rapidly. Subsequent adenosine receptor stimulation induces various effects, including vasodilation, which can protect the tissue against the ischaemic insult. Adenosine deaminase (ADA) is an enzyme that catalyzes the irreversible deamination of adenosine. We hypothesized that the 22G>A polymorphism in the ADA gene inhibits its catalytic activity, and potentiates the protective effects of adenosine. Methods In 96 healthy volunteers, blood was drawn to determine the ADA genotype, and the Vmax and Km values of the ADA from isolated erythrocytes. In a subgroup of volunteers (n=40) we measured the forearm vasodilator response to 13 min of forearm ischaemia using venous occlusion plethysmography as a read-out parameter for adenosine receptor stimulation. Results Although healthy volunteers with the 22GA genotype had a lower Vmax value of ADA than volunteers with the GG genotype (61.6±4.3 ng/min/mg, n=14, vs. 78.0±2.8 ng/min/mg, n=82; P=0.02), this did not potentiate the forearm vasodilator response to 13 min of ischaemia (77.4±8.8 ml/dl in the GA group (n=5) vs. 87.0±5.0 ml/dl (n=35), area under the curve, P=0.3). Conclusion We conclude that heterozygosity for the 22G>A variant of ADA, although reducing catalytic activity, does not enhance forearm reactive hyperaemia. Therefore, the 22G>A variant probably does not contribute to any variability in the protective cardiovascular effects of adenosine.

Dipyridamole augments the antiinflammatory response during human endotoxemia

IntroductionIn animal models of systemic inflammation, the endogenous nucleoside adenosine controls inflammation and prevents organ injury. Dipyridamole blocks the cellular uptake of endogenous adenosine and increases the extracellular adenosine concentration. We studied the effects of oral dipyridamole treatment on innate immunity and organ injury during human experimental endotoxemia.MethodsIn a randomized double-blind placebo-controlled study, 20 healthy male subjects received 2 ng/kg Escherichia coli endotoxin (lipopolysaccharide; LPS) intravenously after 7-day pretreatment with dipyridamole, 200 mg slow release twice daily, or placebo.ResultsNucleoside transporter activity on circulating erythrocytes was reduced by dipyridamole with 89% ± 2% (P < 0.0001), and the circulating endogenous adenosine concentration was increased. Treatment with dipyridamole augmented the LPS-induced increase in the antiinflammatory cytokine interleukin (IL)-10 with 274%, and resulted in a more rapid decrease in proinflammatory cytokines tumor necrosis factor-α (TNF-α) and IL-6 levels directly after their peak level (P < 0.05 and < 0.01, respectively). A strong correlation was found between the plasma dipyridamole concentration and the adenosine concentration (r = 0.82; P < 0.01), and between the adenosine concentration and the IL-10 concentration (r = 0.88; P < 0.0001), and the subsequent decrease in TNF-α (r = -0.54; P = 0.02). Dipyridamole treatment did not affect the LPS-induced endothelial dysfunction or renal injury during experimental endotoxemia.ConclusionsSeven-day oral treatment with dipyridamole increases the circulating adenosine concentration and augments the antiinflammatory response during experimental human endotoxemia, which is associated with a faster decline in proinflammatory cytokines.Trial registrationClinicalTrials (NCT): NCT01091571.

Proximal tubular efflux transporters involved in renal excretion of p-cresyl sulfate and p-cresyl glucuronide : Implications for chronic kidney disease pathophysiology

The uremic solutes p-cresyl sulfate (pCS) and p-cresyl glucuronide (pCG) accumulate in patients with chronic kidney disease (CKD), and might contribute to disease progression. Moreover, retention of these solutes may directly be related to renal tubular function. Here, we investigated the role of the efflux transporters Multidrug Resistance Protein 4 (MRP4) and Breast Cancer Resistance Protein (BCRP) in pCS and pCG excretion, and studied the impact of both solutes on the phenotype of human conditionally immortalized renal proximal tubule epithelial cells (ciPTEC). Our results show that p-cresol metabolites accumulate during CKD, with a shift from sulfation to glucuronidation upon progression. Moreover, pCS inhibited the activity of MRP4 by 40% and BCRP by 25%, whereas pCG only reduced MRP4 activity by 75%. Moreover, BCRP-mediated transport of both solutes was demonstrated. Exposure of ciPTEC to pCG caused epithelial-to-mesenchymal transition, indicated by increased expression of vimentin and Bcl-2, and diminished E-cadherin. This was associated with altered expression of key tubular transporters. In conclusion, BCRP is likely involved in the renal excretion of both solutes, and pCG promotes phenotypical changes in ciPTEC, supporting the notion that uremic toxins may be involved in CKD progression by negatively affecting renal tubule cell phenotype and functionality.

Circulating adenosine increases during human experimental endotoxemia but blockade of its receptor does not influence the immune response and subsequent organ injury

IntroductionPreclinical studies have shown that the endogenous nucleoside adenosine prevents excessive tissue injury during systemic inflammation. We aimed to study whether endogenous adenosine also limits tissue injury in a human in vivo model of systemic inflammation. In addition, we studied whether subjects with the common 34C > T nonsense variant (rs17602729) of adenosine monophosphate deaminase (AMPD1), which predicts increased adenosine formation, have less inflammation-induced injury.MethodsIn a randomized double-blinded design, healthy male volunteers received 2 ng/kg E. Coli LPS intravenously with (n = 10) or without (n = 10) pretreatment with the adenosine receptor antagonist caffeine (4 mg/kg body weight). In addition, lipopolysaccharide (LPS) was administered to 10 subjects heterozygous for the AMPD1 34C > T variant.ResultsThe increase in adenosine levels tended to be more pronounced in the subjects heterozygous for the AMPD1 34C > T variant (71 ± 22%, P=0.04), compared to placebo- (59 ± 29%, P=0.012) and caffeine-treated (53 ± 47%, P=0.29) subjects, but this difference between groups did not reach statistical significance. Also the LPS-induced increase in circulating cytokines was similar in the LPS-placebo, LPS-caffeine and LPS-AMPD1-groups. Endotoxemia resulted in an increase in circulating plasma markers of endothelial activation [intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM)], and in subclinical renal injury, measured by increased urinary excretion of tubular injury markers. The LPS-induced increase of these markers did not differ between the three groups.ConclusionsHuman experimental endotoxemia induces an increase in circulating cytokine levels and subclinical endothelial and renal injury. Although the plasma adenosine concentration is elevated during systemic inflammation, co-administration of caffeine or the presence of the 34C > T variant of AMPD1 does not affect the observed subclinical organ damage, suggesting that adenosine does not affect the inflammatory response and subclinical endothelial and renal injury during human experimental endotoxemia.Trial RegistrationClinicalTrials (NCT): NCT00513110.